GPCR signaling is essential for a wide variety of physiological processes. GPCR signaling can also play an active role in the pathogenesis of multiple diseases. GPCRs are normally tightly regulated so that signals are of the appropriate magnitude and duration and any perturbations in these regulatory processes can be deleterious. Typically, GPCR signaling is tightly regulated by G protein-coupled receptor kinases (GRKs) and beta arrestins. beta arrestins bind to GRK-phosphorylated GPCRs at the plasma membrane to terminate signaling by promoting G protein uncoupling and receptor endocytosis. The molecular mechanisms by which GRKs and beta arrestins regulate GPCR signaling remain poorly understood. The goal of this proposal is to elucidate the molecular mechanisms by which GRKs and beta-arrestins govern GPCR signaling. Recently, we described a novel function for beta arrestins, as endosomal sorting molecules, whereby they function on endosomes to mediate sorting of GPCRs from endosomes to lysosomes, leading to receptor degradation and long-term attenuation of signaling. However, mechanistic insight into this new function is lacking. beta arrestin-1 mediates endosomal sorting of the chemokine receptor CXCR4, which also requires the action of the E3 ubiquitin ligase AIP4 and the ESCRT (endosomal sorting complex required for transport) machinery. How beta arrestin-1 functionally integrates with AIP4 and ESCRTs to mediate CXCR4 endosomal sorting remains to be determined. Based on our strong preliminary data we hypothesize that beta arrestin-1 regulates the ESCRT machinery to control CXCR4 endosomal trafficking and signaling. We propose the following two specific aims: Aim 1: To determine the molecular mechanisms by which beta-arrestin-1 integrates with AIP4 and ESCRTs to control CXCR4 endosomal sorting. Aim 2: To determine the molecular mechanisms by which ESCRTs regulate CXCR4 signaling and function. We will utilize a comprehensive series of state-of-the-art biochemical, molecular and cellular biology and imaging approaches to complete these aims. We anticipate that new knowledge gained from this proposal will lead to the identification of new and innovative approaches to manipulate GPCR signaling to prevent and treat a variety of diseases.